Means and methods for nucleic acid delivery vehicle design and nucleic acid transfer

ABSTRACT

Cells capable of at least, in part, complementing adenovirus E2A function of an adenovirus defective in E2A function. Such cells include a nucleic acid encoding adenovirus E2A or a functional part, derivative and/or analogue thereof, integrated into the cell&#39;s genome. The cell may have E2A nucleic acid derived from a temperature sensitive adenovirus. Methods for producing an adenovirus particle containing an adenovirus vector with a functional deletion of E2A are also disclosed. Such methods involve providing a cell with the functionally deleted adenovirus vector, culturing the cell, and harvesting viral particle. The functional deletion can comprise a deletion of nucleic acid encoding E2A. In such a method, the nucleic acid encoding adenovirus E2A in the cell&#39;s genome has no sequence overlap with the vector leading to replication competent adenovirus and/or to the formation of an adenovirus vector comprising E2A function. In the method, the adenovirus vector may further include a functional deletion of E1-region encoding nucleic acid. Methods for providing cells of an individual with a nucleic acid of interest, without risk of administering simultaneously a replication competent adenovirus vector, comprising administering the individual one of the previously described preparations are also disclosed.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 09/298,745,now U.S. Pat. No. ______, incorporated by reference, which is acontinuation-in-part of U.S. patent application No. 08/793,170 filedMar. 25, 1997, pending, incorporated herein by reference, which is thenational stage filing of PCT/NL96/00244 filed Jun. 14, 1996,incorporated herein by reference, claiming priority from EP 95201611.1filed June 15, 1995 and EP 95201728.3 filed Jun. 26, 1995, all of whichare incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to the field of recombinant DNAtechnology, more in particular to the field of gene therapy.Specifically, the present invention relates to gene therapy usingmaterials derived from adenovirus, in particular human recombinantadenovirus, and relates to novel virus derived vectors and novelpackaging cell lines for vectors based on adenoviruses. Furthermore,this invention also pertains to the screening of replication-competentand revertant E1 and/or E2A adenoviruses from recombinant adenovirusesused in gene therapy.

BACKGROUND

[0003] The current generation of adenoviral vectors for gene therapycontains deletions of the early region 1 (“E1”), where new geneticinformation can be introduced. The E1 deletion renders the recombinantvirus replication defective. It was generally thought that E1-deletedvectors would not express any other adenoviral genes, because E1 isreported to trigger the transcription of the other adenoviral genes. Ithas been shown by us and others that these vectors express several early(e.g., E2A) and late genes (e.g., fiber and penton-base) in the absenceof E1. This means that delivery of a therapeutic gene using E1-deletedadenoviral vectors will result in expression of the therapeutic proteinand adenoviral proteins. A cytotoxic immune response is evoked againstsuch transduced cells. It has been shown that cytotoxic T-lymphocytes(“CTLs”) directed against both the transgene product and productsencoded by the vector are activated, following vector administrationinto immunocompetent animals (Song et al., Hum. Gene Ther. 8: 1207,1997; Yang et al., J. Virol. 70: 7209, 1996). Activated CTLssubsequently eradicate transduced cells from the recipient. Consistentwith this, the longevity of transgene expression is significantlyextended in immuno-deficient and immuno-compromised animals.

[0004] Expression of at least some adenoviral genes in a target cell isat least in part due to background replication of the recombinantadenoviral vector genome and/or background activity of promoters drivingthe respective adenoviral genes (Yang et al., Nature Genet. 7: 362,1994; Lusky et al., J. Virol. 72: 2022, 1998). As a result of theexpression of at least some adenovirus proteins in a target cell in arecipient, an immune response may be mounted against transduced cells.Such an immune response is often not desired, especially when long-termexpression of a transgene is aimed for. One mechanism by whichadenovirus proteins in a target cell in a recipient may cause the immunesystem of the recipient to remove the target cell is the following.Proteins encoded by expressed adenovirus genes can be processed intosmall peptides in a proteosome of the target cell. Peptides producedduring this processing can subsequently be presented at the cell surfaceof the transduced cells in the complex of MHC class-I andβ2-microglobulin molecules. Finally, one or more of the peptides may berecognized as non-self peptides by circulating CTLs whereupon transducedcells can be eradicated from the recipient (reviewed in Ploegh, Science280: 248, 1998).

DISCLOSURE OF THE INVENTION

[0005] In one aspect the present invention provides at least in part asolution to the problem of undesired removal of target cells in arecipient.

[0006] The present invention also provides, at least in part, a solutionfor the immune response against viral proteins. To this end, theinvention provides improved recombinant adenoviral vectors that, inaddition to deletion of E1, are also deleted for the adenoviral early 2Agene (“E2A gene” or “E2A”). The protein encoded by E2A is expressed fromrecombinant E1-deleted adenoviral vectors. In addition to that, residualexpression of E2A from E1-deleted recombinant adenoviral vectors inducesthe expression of the viral late genes, since DNA binding protein(“DBP”) has a positive regulatory effect on the adenovirus major latepromoter (“MLP”) and, therefore, on the expression of the late genes(Chang et al., J Virol. 64: 2103, 1990). Deletion of the E2A gene fromthe recombinant adenoviral genome will therefore improve thecharacteristics of recombinant adenoviral vectors. First, deletion ofE2A will eliminate the synthesis of DBP. Second, it will inhibit thebackground replication of the recombinant adenoviral backbone. Third, itwill reduce the residual expression of the late genes. Finally, it willincrease the capacity of the vector to harbor larger and/or multipletransgenes.

[0007] The E2A gene encodes the 72-kDa protein single stranded DBP whoseactivity is pivotal for the adenovirus DNA replication (reviewed in TheMolecular Repertoire of Adenoviruses II, Springer-Verlag 1995).Therefore, manufacturing of vectors that are deleted for E2A requires acell line that complements for the deletion of E2A in the recombinantadenoviral vector. Major hurdles in this approach are:

[0008] a) that E2A should be expressed to very high levels and

[0009] b) that constitutive expression of E2A is toxic for cells and,therefore, impossible to achieve (Klessig et al., Mol. Cell Biol. 4:1354, 1984).

[0010] The current invention, therefore, involves the use of atemperature sensitive mutant of E2A derived from a temperature sensitiveadenovirus under control of strong viral enhancer sequences, e.g., thecytomegalovirus enhancer for the generation of E2A complementing celllines. DBP (ts125E2A) from hAd5ts125 is inactive at 39° C., but is fullyactive at 32° C. High levels of this protein can be maintained in thenew complementing cells of the invention at the non-permissivetemperature, until the switch is made to the permissive temperature. Theinvention also provides means and methods to use the complementing cellline, comprising E2A, tsE2A, or both E1 and tsE2A, for the generation ofE2A- or E1- and E2A-deleted adenoviral vectors. The invention alsoinvolves inducible expression of E2A or tsE2A.

[0011] The invention also provides new cell lines that complement forE2A or for both the E1 and the E2A deletion in the vector. The inventionalso provides new recombinant adenoviral vectors deleted for E2A or bothE1 and E2A.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 depicts the temperature dependent growth of PER.C6. PER.C6cells were cultured in Dulbecco's Modified Eagle Medium supplementedwith 10% Fetal Bovine Serum (FBS, Gibco BRL) and 10 mM MgCl₂ in a 10%CO₂ atmosphere at either 32° C., 37° C. or 39° C. At a total of 1×10⁶PER.C6 cells were seeded per 25 cm² tissue culture flask (Nunc) and thecells were cultured at either 32° C., 37° C. or 39° C. At each of days1-8, cells were counted. The growth rate and the final cell density ofthe PER.C6 culture at 39° C. are comparable to that at 37° C. The growthrate and final density of the PER.C6 culture at 32° C. were slightlyreduced as compared to that at 37° C. or 39° C.

[0013]FIG. 2 depicts DBP levels in PER.C6 cells transfected with pcDNA3,pcDNA3wtE2A or pcDNA3ts125E2A. Equal amounts of whole-cell extract werefractionated by SDS-PAGE on 10% gels. Proteins were transferred ontoImmobilon-P membranes and DBP protein was visualized using the aDBPmonoclonal B6 in an ECL detection system. All of the cell lines derivedfrom the pcDNA3ts 125E2A transfection express the 72-kDa E2A-encoded DBPprotein (left panel, lanes 4-14; middle panel, lanes 1-13; right panel,lanes 1-12). In contrast, the only cell line derived from the pcDNAwtE2Atransfection did not express the DBP protein (left panel, lane 2). NoDBP protein was detected in extract from a cell line derived from thepcDNA3 transfection (left panel, lane 1), which serves as a negativecontrol. Extract from PER.C6 cells transiently transfected withpcDNA3ts125 (left panel, lane 3) served as a positive control for theWestern blot procedure. These data confirm that constitutive expressionof wtE2A is toxic for cells and that using the ts125 mutant of E2A cancircumvent this toxicity.

[0014]FIG. 3 depicts DBP expression in pcDNA3ts125E2A transfected 293cells. Equal amounts of whole-cell extract were fractionated by SDS-PAGEon 10% gels. Proteins were transferred onto Immobilon-P membranes andDBP protein was visualized using the aDBP monoclonal B6 in an ECLdetection system. Clone 20 (lane 8) from the pcDNA3ts125E2A transfected293 cells expressed the full-length ts125E2A encoded 72-kDa DBP. No E2Aencoded DBP was detected in the extract from a cell line (clone 4)derived from the pcDNA3 transfected 293 cells (lane 1), which serves asa negative control. Extract from PER.C6 cells stably expressing ts125E2Aencoded DBP (polyclonal cell line 5) (lane 2) served as a positivecontrol for the Western blot procedure. The other 293 clones either didnot express ts125E2A encoded DBP (clones 21 and 22, lanes 9 and 10respectively) or expressed aberrant products running with a faster(clones 3, 12, 16 and 18, lanes 4-7) or slower (clone 2, lane 3)mobility in SDS/PAGE.

[0015]FIG. 4 depicts suspension growth of PER.C6ts125E2A cell line c5-9.PER.C6ts125E2Ac5-9 cells were seeded in a 125ml tissue cultureErlenmeyer at a seeding density of 3×10⁵ cells per ml in a total volumeof 20 ml serum-free medium. Cells were further cultured at 125 RPM on anorbital shaker at 39° C. in a 10% CO₂ atmosphere. Cells were counted ateach of days 1-6. The mean growth curve from 8 cultures is shown.PER.C6ts125E2Ac5-9 performs well in serum-free suspension culture. Themaximum cell density of approximately 2×10⁶ cells per ml is reachedwithin 5 days of culture.

[0016]FIG. 5 depicts growth curve PER.C6 and PER.C6tsE2A. PER.C6 cellsor PER.C6ts125E2A (c8-4) cells were cultured at 37° C. or 39° C.,respectively. At day 0, a total of 1×10⁶ cells was seeded per 25 cm²tissue culture flask. At the indicated time points, cells were counted.The growth of PER.C6 cells at 37° C. is comparable to the growth ofPER.C6ts125E2A c8-4 at 39° C. This shows that constitutiveover-expression of ts125E2A has no adverse effect on the growth of cellsat the non-permissive temperature of 39° C.

[0017]FIG. 6 depicts stability of PER.C6ts125E2A. For several passages,the PER.C6ts125E2A cell line clone 8-4 was cultured at 39° C. in mediumwithout G418. Equal amounts of whole-cell extract from different passagenumbers were fractionated by SDS-PAGE on 10% gels. Proteins weretransferred onto Immobilon-P membranes and DBP protein was visualizedusing the aDBP monoclonal B6 in an ECL detection system. The expressionof ts125E2A encoded DBP is stable for at least 16 passages, which isequivalent to approximately 40 cell doublings. No decrease in DBP levelswas observed during this culture period, indicating that the expressionof ts125E2A is stable, even in the absence of G418 selection pressure.

[0018]FIG. 7 depicts revertant-free manufacturing of DE1/E2A vectors.The recombinant adenoviral vector DNA was screened for reversion of theE2A deleted phenotype by PCR. As shown in the left panel, E2A sequenceswere amplified from the DNA samples (+) and control samples (−) spikedwith both 1, 10 and 40 molecules using primer set A, as evidenced by theamplification of a 260 base pair (“bp”) DNA fragment. In contrast, noE2A sequences were amplified from the non-spiked samples, showing thatreversion of the E2A-deleted did not occur. As shown in the right panel,the PCR reactions with primer set B yielded the expected DNA fragment of169 bp in the samples containing the recombinant adenoviral vector DNA(+). From the negative control samples containing the water instead ofDNA (−), no DNA fragment of 169 bp was amplified. These data show thatelimination of overlap between adenoviral sequences in the vector andcell line prevents reversion of the E2A-deleted phenotype.

[0019]FIG. 8 depicts transduction of HeLa cells with IG.Ad/CMV.LacZ andIG.Ad/CMV.LacZDE2A. HeLa cells were infected with a multiplicity ofinfection (“m.o.i.”) of either 0, 10, 100 or 1000 viral particlesIG.Ad/CMV.LacZ or IG.Ad/CMV.LacZDE2A per cell. Forty-eight hours postinfection, cells were stained with X-gal solution. IG.Ad/CMV.LacZDE2Atransduced HeLa cells stained at least as good as did IG.Ad/CMV.LacZ, atall m.o.i.'s.

[0020]FIG. 9 depicts luciferase activity in infected A549 and HeLacells. HeLa and A549 cells were infected with a m.o.i. of either 0, 10,100, 1,000 or 10,000 virus particles (“vp”) IG.Ad/CLIP.Luc orIG.Ad/CLIP.LucDE2A per cell. Two days post infection, cells were lysedand the luciferase activity was determined. Both the IG.Ad/CLIP.LucDE2Ainfected HeLa and A549 cells produce more luciferase enzyme than theIG.Ad/CLIP.Luc infected HeLa and A549 and HeLa cells, at all m.o.i.'stested.

[0021]FIG. 10 depicts the expression of DBP, Penton and Fiber. A549cells were infected with a m.o.i. of either 0, 100, 1,000 or 10,000vp/cell IG.Ad/CLIP or IG.Ad.CLIPDE2A. Seventy-two hours post infection,cell extracts were prepared and equal amounts of whole cell extract werefractionated by SDS-PAGE on 10% gels. The proteins were visualized withthe aDBP monoclonal B6, the polyclonal a-Penton base Ad2-Pb571 or thepolyclonal a-knob domain of fiber E641/3, using an ECL detection system.Cells infected with IG.Ad.CLIP express both E2A encoded DBP, Penton baseand Fiber proteins. The proteins co-migrate with the respective proteinsin the positive control (lane P, extract from PER.C6 cells infected withIG.Ad.CLIP harvested at starting CPE). In contrast, no DBP, penton-baseor fiber was detected in the non-infected A549 cells or cells infectedwith IG.Ad.CLIPDE2A. These data show that deletion of the E2A gene didnot only eliminate residual DBP expression, but also the residualexpression of the late adenoviral proteins penton-base and fiber.

Best Mode of The Invention

[0022] According to a presently preferred embodiment of the invention, acell according to the invention is capable of at least, in part,complementing adenovirus E2A function of an adenovirus defective in E2Afunction. Such a cell includes a nucleic acid encoding adenovirus E2A ora functional part, derivative and/or analogue thereof, integrated intothe genome of the cell. Preferably, the cell has E2A nucleic acidderived from a temperature sensitive adenovirus such as but not limitedto adenovirus ts125. More preferably, such a cell further includes anucleic acid encoding adenovirus E1-region proteins or a functionalpart, derivative and/or analogue thereof. Such a cell could be derivedfrom the “PER.C6” cell line (commercially available from IntroGene, by,and deposited, under ECACC deposit accession number 96022940 under theprovisions of the Budapest Treaty with the Centre for AppliedMicrobiology and Research Authority (European Collection of Animal CellCultures), Porton Down, Salisbury, Wiltshire SP4, OJG, United Kingdom,an International Depository Authority, in accordance with the BudapestTreaty, on Feb. 29, 1996.

[0023] The invention also includes a method for producing an adenovirusparticle containing an adenovirus vector with a functional deletion ofE2A. Such a method involves providing a cell as previously describedwith the functionally deleted adenovirus vector, culturing the cell, andharvesting the virus particle. In such a method, the functional deletioncan comprise a deletion of at least part of the nucleic acid encodingE2A. In such a method, the nucleic acid encoding adenovirus E2A in thegenome of the cell preferably has no sequence overlap with the vectorwhich leads to replication competent adenovirus and/or to the formationof an adenovirus vector comprising E2A function. In the method, theadenovirus vector preferably further comprises a functional deletion ofE1-region encoding nucleic acid, comprising providing one of thepreviously described cells with the adenovirus vector, culturing thecell and harvesting the virus particle. In such a method, the nucleicacid encoding adenovirus E1-region preferably does not comprise sequenceoverlap with the vector which leads to replication competent adenovirusand/or to the formation of an adenovirus vector comprising an E1function. Furthermore, in the method, the adenovirus vector furthercomprises at least one nucleic acid of interest.

[0024] The invention also includes an adenovirus vector comprising afunctional deletion of adenovirus E2A. Such a functional deletion ispreferably a deletion of at least part of the nucleic acid encoding E2A.The deletion may encompass the entire coding region of E2A. Such anadenovirus vector preferably includes a deletion corresponding to adeletion of nucleotides 22443 to 24032 in adenovirus 5. The deletion caninclude a deletion of nucleic acid encoding E1-region proteins. Thedeletion of nucleic acid encoding E1-region proteins can comprise adeletion corresponding to a deletion of nucleotides 459 to 3510 inadenovirus 5. Again, the adenovirus vector preferably further includesat least one nucleic acid of interest.

[0025] An adenovirus vector according to the invention can, but does notnecessarily, also comprise at least a deletion of a region which inadenovirus 5 corresponds to nucleotides 22418-24037 or a deletion of aregion which in adenovirus 5 corresponds to nucleotides 22443-24032.Such vectors can further comprise at least nucleic acid which inadenovirus 5 corresponds to nucleotides 3534-22347 and/or nucleotides24060 until the right ITR or at least 3534-22417 and/or 24038 until theright ITR or at least nucleic acid which in adenovirus 5 corresponds tonucleotides 3534-22442 and/or nucleotides 24033 until the right ITR.

[0026] The invention also includes preparations of adenovirus vectorcontaining adenovirus particles wherein the adenovirus vector comprisesa functional deletion of E2A. Such an adenovirus vector preferablyfurther includes a deletion of nucleic acid encoding E1-region proteins,and may be free of adenovirus vectors comprising E2A function. In such acase, the preparation may be free of adenovirus vectors comprisingnucleic acid encoding a functional E2A, or a functional part, derivativeand/or analogue thereof. The preparation is preferably free ofadenovirus vectors comprising nucleic acid encoding E1-region proteinsor parts, derivatives and/or analogues thereof.

[0027] The invention also includes a method for providing cells of anindividual with a nucleic acid of interest, without risk ofadministering simultaneously a replication competent adenovirus vector,comprising administering the individual one of the previously describedpreparations.

[0028] The invention is further described by the use of the followingillustrative Examples.

EXAMPLE I

[0029] Generation of producer cell lines for the production ofrecombinant adenoviral vectors deleted in E1 and E2A or E1 and E2A

[0030] Here is described the generation of cell lines for the productionof recombinant adenoviral vectors that are deleted in E1 and E2A. Theproducer cell lines complement for the E1 and E2A deletion fromrecombinant adenoviral vectors in trans by constitutive expression ofthe E1 and E2A genes, respectively. The pre-established Ad5-E1transformed human embryo retinoblast cell line PER.C6 (commerciallyavailable from IntroGene, by (now Crucell, NV) of Leiden, NL, see also,International Patent Appln. WO 97/00326) and Ad5 transformed humanembryo kidney cell line 293 (Graham et al., J Gen. Virol. 36: 59, 1977)were further equipped with E2A expression cassettes.

[0031] The adenoviral E2A gene encodes a 72 kDa DBP which has a highaffinity for single stranded DNA. Because of its function, constitutiveexpression of DBP is toxic for cells. The ts125E2A mutant encodes a DBPwhich has a Pro→Ser substitution of amino acid 413 (van der Vliet, JVirol. 15: 348, 1975). Due to this mutation, the ts125E2A encoded DBP isfully active at the permissive temperature of 32° C., but does not bindto ssDNA at the non-permissive temperature of 39° C. This allows thegeneration of cell lines that constitutively express E2A, which is notfunctional and is not toxic at the non-permissive temperature of 39° C.Temperature sensitive E2A gradually becomes functional upon temperaturedecrease and becomes fully functional at a temperature of 32° C., thepermissive temperature.

[0032] A. Generation of Plasmids Expressing the Wild Type E2A—orTemperature Sensitive ts125E2A Gene.

[0033] pcDNA3wtE2A: The complete wild-type E2A coding region wasamplified from the plasmid pBR/Ad.Bam-rITR (ECACC deposit P97082122)with the primers DBPpcr1 and DBPpcr2 using the Expand™ Long Template PCRsystem according to the standard protocol of the supplier (BoehringerMannheim). The PCR was performed on a Biometra TRIO THERMOBLOCK, usingthe following amplification program: 94° C. for 2 minutes, 1 cycle; 94°C. for 10 seconds+51° C. for 30 seconds+68° C. for 2 minutes, 1 cycle;94° C. for 10 seconds+58° C. for 30 seconds+68° C. for 2 minutes, 10cycles; 94° C. for 10 seconds+58° C. for 30 seconds+68° C. for 2 minuteswith 10 seconds extension per cycle, 20 cycles; 68° C. for 5 minutes, 1cycle. The primer DBPpcr1: CGG GAT CCG CCA CCA TGG CCA GTC GGG AAG AGGAG (5′ to 3′) (SEQ ID NO:1) contains a unique BamHI restriction site(underlined) 5′ of the Kozak sequence (italic) and start codon of theE2A coding sequence. The primer DBPpcr2: CGG AAT TCT TAA AAA TCA AAG GGGTTC TGC CGC (5′ to 3′) (SEQ ID NO:2) contains a unique EcoRI restrictionsite (underlined) 3 ′ of the stop codon of the E2A coding sequence. Thebold characters refer to sequences derived from the E2A coding region.The PCR fragment was digested with BamHI/EcoRI and cloned intoBamHI/EcoRI digested pcDNA3 (Invitrogen), giving rise to pcDNA3wtE2A.

[0034] pcDNA3tsE2A: The complete ts125E2A-coding region was amplifiedfrom DNA isolated from the temperature sensitive adenovirus mutantH5ts125 (Ensinger et al., J. Virol. 10: 328, 1972; van der Vliet et al.,J Virol. 15: 348, 1975). The PCR amplification procedure was identicalto that for the amplification of wtE2A. The PCR fragment was digestedwith BamHI/EcoRI and cloned into BamHI/EcoRI digested pcDNA3(Invitrogen), giving rise to pcDNA3tsE2A. The integrity of the codingsequence of wtE2A and tsE2A was confirmed by sequencing.

[0035] B. Growth Characteristics of Producer Cells for the Production ofRecombinant Adenoviral Vectors Cultured at 32°, 37° and 39° C.

[0036] PER.C6 cells were cultured in Dulbecco's Modified Eagle Medium(“DMEM”, Gibco BRL) supplemented with 10% FBS and 10 mM MgCl₂ in a 10%CO₂ atmosphere at either 32° C., 37° C. or 39° C. At day 0, a total of1×10⁶ PER.C6 cells were seeded per 25 cm² tissue culture flask (Nunc)and the cells were cultured at either 32° C., 37° C. or 39° C. At eachof days 1-8, cells were counted. FIG. 1 shows that the growth rate andthe final cell density of the PER.C6 culture at 39° C. are comparable tothat at 37° C. The growth rate and final density of the PER.C6 cultureat 32° C. were slightly reduced as compared to that at 37° C or 39° C.No significant cell death was observed at any of the incubationtemperatures. Thus PER.C6 performs very well both at 32° C. and 39° C.,the permissive and non-permissive temperature for ts125E2A,respectively.

[0037] C. Transfection of PER.C6 and 293 With E2A Expression Vectors;Colony Formation and Generation of Cell Lines.

[0038] One day prior to transfection, 2×10⁶ PER.C6 cells were seeded per6 cm tissue culture dish (Greiner) in DMEM, supplemented with 10% FBSand 10 mM MgCl₂ and incubated at 37° C. in a 10% CO₂ atmosphere. Thenext day, the cells were transfected with 3, 5 or 8μg of either pcDNA3,pcDNA3wtE2A or pcDNA3tsE2A plasmid DNA per dish, using the LipofectAMINEPLUS™ Reagent Kit according to the standard protocol of the supplier(Gibco BRL), except that the cells were transfected at 39° C. in a 10%CO₂ atmosphere. After the transfection, the cells were constantly keptat 39° C., the non-permissive temperature for ts125E2A. Three dayslater, the cells were put on DMEM, supplemented with 10% FBS, 10 mMMgCl₂ and 0.25 mg/ml G418 (Gibco BRL) and the first G418 resistantcolonies appeared at 10 days post transfection. As shown in Table 1,there was a dramatic difference between the total number of coloniesobtained after transfection of pcDNA3 (˜200 colonies) or pcDNA3tsE2A(˜100 colonies) and pcDNA3wtE2A (only 4 colonies). These resultsindicate that the constitutive expression of E2A is toxic and thetoxicity of constitutively expressed E2A can be overcome by using atemperature sensitive mutant of E2A (ts125E2A) and culturing of thecells at the non-permissive temperature of 39° C. TABLE 1 Number ofcolonies after transfection of PER.C6 with E2A expression vectors:plasmid number of colonies cell lines established pcDNA3 ˜200 4/4PcDNA3wtE2A 4 1/4 PcDNA3tsE2A ˜100 37/45

[0039] PER.C6 cells were transfected with either pcDNA3, pcDNA3wtE2A orpcDNA3wtE2A and cultured in selection medium containing 0.25 mg/ml G418at 39° C. All colonies (4/4) picked from the pcDNA3 transfection and 82%(37/45) of the colonies from the pcDNA3tsE2A transfection wereestablished to stable cell lines. In contrast, only 25% (1/4) of thecolonies from the pcDNA3wtE2A transfection could be established to acell line.

[0040] From each transfection, a number of colonies was picked byscraping the cells from the dish with a pipette. The detached cells weresubsequently put into 24 well tissue culture dishes (Greiner) andcultured further at 39° C. in a 10% CO₂ atmosphere in DMEM, supplementedwith 10% FBS, 10 mM MgCl₂ and 0.25mg/ml G418. As shown in Table 1, 100%of the pcDNA3 transfected colonies (4/4) and 82% of the pcDNA3tsE2Atransfected colonies (37/45) were established to stable cell lines (theremaining 8 pcDNA3tsE2A transfected colonies grew slowly and werediscarded). In contrast, only 1 pcDNA3wtE2A-transfected colony could beestablished. The other 3 died directly after picking.

[0041] Next, the E2A expression levels in the different cell lines weredetermined by Western blotting. The cell lines were seeded on 6 welltissue culture dishes and sub-confluent cultures were washed twice withPBS (NPBI) and lysed and scraped in RIPA (1% NP-40, 0.5% sodiumdeoxycholate and 0.1% SDS in PBS, supplemented with 1 mMphenylmethylsulfonylfluoride and 0.1 mg/ml trypsin inhibitor). After 15minutes incubation on ice, the lysates were cleared by centrifugation.Protein concentrations were determined by the Bio-Rad protein assay,according to standard procedures of the supplier(BioRad). Equal amountsof whole-cell extractwere fractionated by SDS-PAGE on 10% gels. Proteinswere transferred onto Immobilon-P membranes (Millipore) and incubatedwith the aDBP monoclonal antibody B6 (Reich et al., Virology 128: 480,1983). The secondary antibody was a horseradish-peroxidase-conjugatedgoat anti mouse antibody (BioRad). The Western blotting procedure andincubations were performed according to the protocol provided byMillipore. The complexes were visualized with the ECL detection systemaccording to the manufacturer's protocol (Amersham). FIG. 2 shows thatall of the cell lines derived from the pcDNA3tsE2A transfection expressthe 72-kDa E2A protein (left panel, lanes 4-14; middle panel, lanes 1-13; right panel, lanes 1- 12). In contrast, the only cell line derivedfrom the pcDNAwtE2A transfection did not express the E2A protein (leftpanel, lane 2). No E2A protein was detected in extract from a cell linederived from the pcDNA3 transfection (left panel, lane 1), which servesas a negative control. Extract from PER.C6 cells transiently transfectedwith pcDNA3ts125 (left panel, lane 3) served as a positive control forthe Western blot procedure. These data confirm that constitutiveexpression of wtE2A is toxic for cells and that using the ts125 mutantof E2A can circumvent this toxicity.

[0042] In contrast to PER.C6 cells, the culturing of 293 cells at 39° C.is troublesome. Therefore, the transfection of 293 cells with eitherpcDNA3, pcDNA3wtE2A or pcDNA3tsE2A was performed at 37° C. in anatmosphere of 10% CO₂, a semi-permissive temperature for ts125E2Aencoded DBP. A day prior to transfection, 293 cells were seeded in DMEM,supplemented with 10% FBS and 10 MM MgCl₂, at a density of 3.6×10⁵ cellsper 6 cm tissue culture dish (Greiner). Five hours before transfection,cells received fresh medium. Cells were transfected with 7.2 μg ofeither pcDNA3, pcDNA3wtE2A or pcDNA3tsE2A plasmid DNA using the CalciumPhosphate Transfection System according to the standard protocol of thesupplier (Gibco BRL). Two days post transfection, cells were put onselection medium, i.e., DMEM supplemented with 10% FBS, 10 mM MgCl₂ and0.1 mg/ml G418. The first colonies appeared at day 12 post transfection.As shown in Table 2, the total number of colonies obtained aftertransfection of pcDNA3 (18 100 colonies) or pcDNA3tsE2A (˜25 colonies)was significantly higher than that obtained after transfection ofpcDNA3wtE2A (only 2 colonies). A total of 22 clones from the pcDNA3tsE2Atransfection were picked by scraping the cells from the dish with apipette. The detached cells were subsequently put into 96 well tissueculture dishes (Greiner) and cultured further at 37° C. in a 10% CO₂atmosphere in DMEM, supplemented with 10% FBS, 10 mM MgCl₂ and 0.1 mg/mlG418. Sixteen out of the 22 picked colonies could be established as celllines (the 6 remaining colonies grew badly and were discarded). TABLE 2Number of colonies after transfection of 293 with E2A expressionvectors: plasmid number of colonies pcDNA3 ˜100 PcDNA3wtE2A 2PcDNA3tsE2A 25

[0043] Selection of colonies derived from 293 cells transfected with E2Aexpression cassettes. Cell line 293 was transfected with either pcDNA3,pcDNA3wtE2A or pcDNA3wtE2A and cultured in selection medium containing0.1 mg/ml G418 at 37° C.

[0044] Next, the E2A expression level in 8 different cell lines wasdetermined by Western blotting. The cell lines were seeded on 6 welltissue culture dishes and sub-confluent cultures were washed twice withPBS (NPBI) and lysed and scraped in RIPA (1% NP-40, 0.5% sodiumdeoxycholate and 0.1% SDS in PBS, supplemented with 1 mMphenylmethylsulfonylfluoride and 0.1 mg/ml trypsin inhibitor). After 15minutes incubation on ice, the lysates were cleared by centrifugation.Protein concentrations were determined by the BioRad protein assay,according to standard procedures of the supplier (BioRad). Equal amountsof whole-cell extract were fractionated by SDS-PAGE on 10% gels.Proteins were transferred onto Immobilon-P membranes (Millipore) andincubated with the aDBP monoclonal antibody B6 (Reich et al., Virology128: 480, 1983). The secondary antibody was ahorseradish-peroxidase-conjugated goat anti mouse antibody (BioRad). TheWestern blotting procedure and incubations were performed according tothe protocol provided by Millipore. The complexes were visualized withthe ECL detection system according to the manufacturer's protocol(Amersham). FIG. 3 shows, that, in contrast to the PER.C6tsE2A celllines, only clone 20 (lane 8) from the pcDNA3tsE2A transfected 293 cellsexpressed the full-length ts125E2A encoded 72-kDa DBP. No E2A encodedDBP was detected in extract from a cell line (clone 4) derived from thepcDNA3 transfected 293 cells (lane 1), which serves as a negativecontrol. Extract from PER.C6 cells stably expressing ts125E2A encodedDBP (polyclonal cell line 5) (lane 2) served as a positive control forthe Western blot procedure. The other 293 clones either did not expressts125E2A encoded DBP (clones 21 and 22, lanes 9 and 10 respectively) orexpressed aberrant products running with a faster (clones 3, 12, 16 and18 lanes 4-7) or slower (clone 2, lane 3) mobility in SDS/PAGE. Theseresults show that generation of E2A complementing cell line by usingtemperature sensitive mutants of E2A is not specific for PER.C6 cells,but that it applies to eukaryotic cells in general (e.g., 293 cells). Inaddition, the 293 data show that keeping the temperature sensitive E2Aencoded DBP as inactive as possible is crucial for easy generation ofsuch cell lines. The 293 cell lines were generated at an intermediatetemperature of 37° C., a temperature at which ts125E2A encoded DBP isonly partially inactivated. This explains the high number of cell linesexpressing aberrant DBP products.

[0045] D. Complementation of E2A Deletion in Adenoviral Vectors onPER.C6- and 293 Cells Constitutively Expressing Full-length ts125E2AEncoded DBP.

[0046] The adenovirus Ad5.dl802 is an Ad 5 derived vector deleted forthe major part of the E2A coding region and does not produce functionalDBP (Rice et al., J Virol. 56: 767, 1985). Ad5.dl802 was used to testthe E2A trans-complementing activity of PER.C6 cells constitutivelyexpressing ts125E2A. Parental PER.C6 cells or PER.C6tsE2A clone 3-9 werecultured in DMEM, supplemented with 10% FBS and 1 mM MgCl₂ at 39° C. . d10% CO₂ in 25 cm² flasks and either mock infected or infected withAd5.dl802 at an m.o.i. of 5. Subsequently, the infected cells werecultured at 32° C. and cells were screened for the appearance of acytopathic effect (CPE) as determined by changes in cell morphology anddetachment of the cells from the flask. Table 3 shows that full CPEappeared in the Ad5.dl802 infrected PER.C6tsE2A clone 3-9 within 2 days.No CPE appeared in the Ad5.dl802 infected PER.C6 cells or the mockinfected cells. These data show that PER.C6 cells constitutivelyexpresing ts125E2A complement in trans for the E2A deletion in theAd5.dl802 vector at the permissive temperature of 32° C.

[0047] These cells are therefore suitable for production of recombinantadenoviral vector that are deficient for functional E2A. TABLE 3Complementation of E2A deletion in adenoviral vectors on PER.C6 cellsand PER.C6 cells constitutively expressing temperature sensitive E2A.32° C. day 2 PER.C6 mock — PER.C6 d1802 — PER.C6ts125c3-9 mock —PER.C6ts125c3-9 d1802 Full CPE

[0048] Parental PER.C6 cells or PER.C6ts125E2A clone 3-9 were infectedwith Ad5.dl802, an Ad5 adenovirus deleted for the E2A gene, at m.o.i. of5. Subsequently, the infected cells were cultured at 32° C. and cellswere screened for the appearance of a cytopathic effect (CPE) asdetermined by changes in cell morphology and detachment of the cellsfrom the flask.

[0049] The 293tsE2A clones c2, c16, c18 and c20 and the 293pcDNA3-clonec4 were tested for their E2A trans-complementing activity as follows.The cell lines were cultured in DMEM, supplemented with 10% FBS and 10mM MgCl₂ at 39° C. and 10% CO₂ in 6 well plates and either mock infectedor infected with IG.Ad.CLIP.Luc (see below) at an m.o.i. of 10.Subsequently, the infected cells were cultured at either 32° C. or 39°C. and cells were screened for the appearance of a cytopathic effect(CPE) 3 days post infection, as determined by changes in cell morphologyand detachment of the cells from the flask. Table 4 shows that no CPEappeared in the control cell line 293pcDNA3-c4. Moreover, the cell linesexpressing aberrant forms of DBP either failed to complement this vector(clones 16 and 18) or were intermediate in the trans-complementingability (clone 2). Only the 293 cell line expressing full-lengthts125E2A encoded DBP (i. e., clone 20) fully complemented for the E2Adeletion in the vector IG.Ad.CLIP.Luc at the permissive temperature of32° C. No CPE appeared at the non-permissive temperature of 39° C. TABLE4 Complementation of E2A deletion in adenoviral vectors on 293 cells and293 cells constitutively expressing temperature sensitive E2A. Cell lineCPE at 32° C. CPE at 39° C. 293pcDNA3-c4 − − 293ts125E2A-c2 +/− −293ts125E2A-c16 − − 293ts125E2A-c18 − − 293ts125E2A-c20 + −

[0050] The 293ts125E2A clones c2, c16, c18 and c20 and the293pcDNA3-clone c4 were tested for their E2A trans-complementingactivity as follows. The cell lines were either mock infected orinfected with IG.Ad.CLIP.Luc at an m.o.i. of 10. Subsequently, theinfected cells were cultured at either 32° C. or 39° C. and cells werescreened for the appearance of a cytopathic effect (CPE) 3 days postinfection, as determined by changes in cell morphology and detachment ofthe cells from the flask.

[0051] E. Serum-free Suspension Culture of PER.C6tsE2A Cell Lines.

[0052] Large-scale production of recombinant adenoviral vectors forhuman gene therapy requires an easy and scalable culturing method forthe producer cell line, preferably a suspension culture in medium devoidof any human or animal constituents. To that end, the cell linePER.C6tsE2A c5-9 (designated c5-9) was cultured at 39° C. and 10% CO₂ ina 175 cm² tissue culture flask (Nunc) in DMEM, supplemented with 10% FBSand 10mM MgCl₂. At sub-confluency (70-80% confluent), the cells werewashed with PBS (NPBI) and the medium was replaced by 25 ml serum freesuspension medium Ex-cell™ 525 (JRH) supplemented with 1×L-Glutamin(Gibco BRL), hereafter designated SFM. Two days later, cells weredetached from the flask by flicking and the cells were centrifuged at1000 rpm for 5 minutes. The cell pellet was re-suspended in 5 ml SFM and0.5 ml cell suspension was transferred to an 80 cm² tissue culture flask(Nunc), together with 12 ml fresh SFM. After 2 days, cells wereharvested (all cells are in suspension) and counted in a Burker cellcounter. Next, the cells were seeded in a 125 ml tissue cultureErlenmeyer (Corning) at a seeding density of 3×10⁵ cells per ml in atotal volume of 20 ml SFM. Cells were further cultured at 125 RPM on anorbital shaker (GFL) at 39° C. in a 10% CO₂ atmosphere. Cells werecounted at day 1-6 in a Burker cell counter. In FIG. 4, the mean growthcurve from 8 cultures is shown. PER.C6tsE2A c5-9 performs well in serumfree suspension culture. The maximum cell density of approximately 2×10⁶cells per ml is reached within 5 days of culture.

[0053] F. Growth Characteristics of PER.C6 and PER.C6/E2A at 37° C. and39° C.

[0054] PER.C6 cells or PER.C6ts125E2A (c8-4) cells were cultured in DMEMsupplemented with 10% FBS and 10 mM MgCl₂in a 10% CO₂ atmosphere ateither 37° C. (PER.C6) or 39° C. (PER.C6ts125E2A c8-4). At day 0, atotal of 1×10⁶ cells were seeded per 25 cm² tissue culture flask (Nunc)and the cells were cultured at the respective temperatures. At theindicated time points, cells were counted. FIG. 5 shows that the growthof PER.C6 cells at 37° C. is comparable to the growth of PER.C6ts125E2Ac8-4 at 39° C. This shows that constitutive expression of ts125E2Aencoded DBP has no adverse effect on the growth of cells at thenon-permissive temperature of 39° C.

[0055] G. Stability of PER. C6ts125E2A.

[0056] For several passages, the PER.C6ts125E2A cell line clone 8-4 wascultured at 39° C. and 10% CO₂ in a 25 cm² tissue culture flask (Nunc)in DMEM, supplemented with 10% FBS and 10 mM MgCl₂ in the absence ofselection pressure (G418). At sub-confluency (70-80% confluent), thecells were washed with PBS (NPBI) and lysed and scraped in RIPA (1%NP-40, 0.5% sodium deoxycholate and 0.1 % SDS in PBS, supplemented with1 mM phenylmethylsulfonylfluoride and 0.1 mg/ml trypsin inhibitor).After 15 minutes incubation on ice, the lysates were cleared bycentrifugation. Protein concentrations were determined by the BioRadprotein assay, according to standard procedures of the supplier(BioRad). Equal amounts of whole-cell extract were fractionated bySDS-PAGE on 10% gels. Proteins were transferred onto Immobilon-Pmembranes (Millipore) and incubated with the aDBP monoclonal antibody B6(Reich et al., Virology 128: 480, 1983). The secondary antibody was ahorseradish-peroxidase-conjugated goat anti mouse antibody (BioRad). TheWestern blotting procedure and incubations were performed according tothe protocol provided by Millipore. The complexes were visualized withthe ECL detection system according to the manufacturer's protocol(Amersham). FIG. 6 shows that the expression of ts125E2A encoded DBP isstable for at least 16 passages, which is equivalent to approximately 40cell doublings. No decrease in DBP levels was observed during thisculture period, indicating that the expression of ts125E2A is stable,even in the absence of G418 selection pressure.

EXAMPLE II

[0057] Plasmid based system for the generation of recombinant adenoviralvectors deleted in E1 and E2A

[0058] A. Generation of pBr/Ad.Bam-rITR (ECACC deposit P97082122).

[0059] In order to facilitate blunt end cloning of the inverted terminalrepeat (“ITR”) sequences, wild-type human adenovirus type 5 (Ad5) DNAwas treated with Klenow enzyme in the presence of excess dNTPs. Afterinactivation of the Klenow enzyme and purification by phenol/chloroformextraction followed by ethanol precipitation, the DNA was digested withBamHI. This DNA preparation was used without further purification in aligation reaction with pBr322 derived vector DNA prepared as follows:pBr322 DNA was digested with EcoRV and BamHI, de-phosphorylated bytreatment with TSAP enzyme (Life Technologies) and purified on LMPagarose gel (SeaPlaque GTG). After transformation into competent E.colinDH5a (Life Techn.) and analysis of ampicillin resistant colonies, oneclone was selected that showed a digestion pattern as expected for aninsert extending from the BamHI site in Ad5 to the right ITR.

[0060] Sequence analysis of the cloning border at the right ITR revealedthat the most 3′G residue of the ITR was missing, the remainder of theITR was found to be correct. The missing G residue is complemented bythe other ITR during replication.

[0061] B. Generation of pBr/Ad.Sal-rITR (ECACC Deposit P97082119).

[0062] pBr/Ad.Bam-rITR was digested with BamHI and SalI. The vectorfragment including the adenovirus insert was isolated in LMP agarose(SeaPlaque GTG) and ligated to a 4.8 kb SalI-BamHI fragment obtainedfrom wt Ad5 DNA and purified with the Geneclean II kit (Bio 101, Inc.).One clone was chosen and the integrity of the Ad5 sequences wasdetermined by restriction enzyme analysis. Clone pBr/Ad.Sal-rITRcontains adeno type 5 sequences from the SalI site at bp 16746 up to andincluding the rITR (missing the most 3′ G residue).

[0063] C. pBr/Ad.Cla-Bam (ECACC Deposit P97082117).

[0064] wt Adeno type 5 DNA was digested with ClaI and BamHI, and the20.6-kb fragment was isolated from gel by electro-elution. pBr322 wasdigested with the same enzymes and purified from agarose gel byGeneclean. Both fragments were ligated and transformed into competentDH5a. The resulting clone pBr/Ad.Cla-Bam was analyzed by restrictionenzyme digestion and shown to contain an insert with adenovirussequences from bp 919 to 21566.

[0065] D. Generation of pBr/Ad.AflII-Bam (ECACC Deposit P97082114).

[0066] Clone pBr/Ad.Cla-Bam was linearized with EcoRi (in pBr322) andpartially digested with AflII. After heat inactivation of AflII for 20minutes at 65° C., the fragment ends were filled in with Klenow enzyme.The DNA was then ligated to a blunt double stranded oligo linkercontaining a PacI site (5′-AATTGTCTTAATTAACCGCTTAA-3′ (SEQ ID NO:3)).This linker was made by annealing the following two oligonucleotides:5′-AATTGTCTTAATTAACCGC-3′ (SEQ ID NO:4) and 5′-AATTGCGGTTAATTAAGAC-3 ′(SEQ ID NO:5), followed by blunting with Klenow enzyme. Afterprecipitation of the ligated DNA to change buffer, the ligations weredigested with an excess PacI enzyme to remove concatemers of the oligo.The 22016 bp partial fragment containing Ad5 sequences from bp 3534 upto 21566 and the vector sequences was isolated in LMP agarose (SeaPlaqueGTG), re-ligated and transformed into competent DH5a. One clone that wasfound to contain the PacI site and that had retained the large adenofragment was selected and sequenced at the 5′ end to verify correctinsertion of the PacI linker in the (lost) AflII site.

[0067] E. Generation of pBr/Ad.Bam-rITRpac#2 (ECACC Deposit P97082120)and pBr/Ad.Bam-rITR#8 (ECACC Deposit P97082121).

[0068] To allow insertion of a PacI site near the ITR of Ad5 in clonepBr/Ad.Bam-rITR, about 190 nucleotides were removed between the ClaIsite in the pBr322 backbone and the start of the ITR sequences. This wasdone as follows: pBr/Ad.Bam-rITR was digested with ClaI and treated withnuclease Bal31 for varying lengths of time (2 minutes, 5 minutes, 10minutes and 15 minutes). The extent of nucleotide removal was followedby separate reactions on pBr322 DNA (also digested at the ClaI site),using identical buffers and conditions. Bal31 enzyme was inactivated byincubation at 75° C. for 10 minutes, and the DNA was precipitated andre-suspended in a smaller volume TE buffer. To ensure blunt ends, DNA'swere further treated with T4 DNA polymerase in the presence of excessdNTPs. After digestion of the (control) pBr322 DNA with SalI,satisfactory degradation (˜150 bp) was observed in the samples treatedfor 10 minutes or 15 minutes. The 10 minutes or 15 minutes treatedpBr/Ad.Bam-rITR samples were then ligated to the above-described bluntedPacI linkers (See pBr/Ad.AflII-Bam). Ligations were purified byprecipitation, digested with excess PacI and separated from the linkerson an LMP agarose gel. After re-ligation, DNA's were transformed intocompetent DH5a and colonies analyzed. Ten clones were selected thatshowed a deletion of approximately the desired length and these werefurther analyzed by T-track sequencing (T7 sequencing kit, PharmaciaBiotech). Two clones were found with the PacI linker inserted justdownstream of the rITR. After digestion with PacI, clone #2 has 28 bpand clone #8 has 27 bp attached to the ITR.

[0069] F. Generation of pWE/Ad.AflII-rITR (ECACC Deposit P97082116).

[0070] Cosmid vector pWE15 (Clontech) was used to clone larger Ad5inserts. First, a linker containing a unique PacI site was inserted inthe EcoRI sites of pWE 15 creating pWE.pac. To this end, the doublestranded PacI oligo as described for pBr/Ad.AflII-BamHI was used but nowwith its EcoRI protruding ends. The following fragments were thenisolated by electro-elution from agarose gel: pWE.pac digested withPacI, pBr/AflII-Bam digested with PacI and BamHI and pBr/Ad.Bam-rITR#2digested with Bam-HI and PacI. These fragments were ligated together andpackaged using 1 phage packaging extracts (Stratagene) according to themanufacturer's protocol. After infection into host bacteria, colonieswere grown on plates and analyzed for presence of the complete insert.pWE/Ad.AflII-rITR contains all adenovirus type 5 sequences from bp 3534(AflII site) up to and including the right ITR (missing the most 3′Gresidue).

[0071] G. Generation of pWE/Ad.AflII-EcoRI.

[0072] pWE.pac was digested with ClaI and 5′ protruding ends were filledusing Klenow enzyme. The DNA was then digested with PacI and isolatedfrom agarose gel. pWE/AflII-rITR was digested with EcoRI and aftertreatment with Klenow enzyme digested with PacI. The large 24-kbfragment containing the adenoviral sequences was isolated from agarosegel and ligated to the ClaI-digested and blunted pWE.pac vector usingthe Ligation Express™ kit from Clontech. After transformation ofUltra-competent XL10-Gold cells from Stratagene, clones were identifiedthat contained the expected insert. pWE/AflII-EcoRI contains Ad5sequences from bp 3534-27336.

[0073] H. Generation of pWE/Ad.AflII-rITRDE2A:

[0074] Deletion of the E2A coding sequences from pWE/Ad.AflII-rITR(ECACC deposit P97082116) has been accomplished as follows. Theadenoviral sequences flanking the E2A coding region at the left and theright site were amplified from the plasmid pBr/Ad.Sal.rITR (ECACCdeposit P97082119) in a PCR reaction with the Expand PCR system(Boehringer) according to the manufacturer's protocol. The followingprimers were used: Right flanking sequences (corresponding Ad5nucleotides 24033 to 25180):

[0075] DE2A.SnaBI: 5′-GGC GTA CGT AGC CCT GTC GAA AG-3′ (SEQ ID NO:6)

[0076] DE2A.DBP-start: 5′-CCA ATG CAT TCG AAG TAC TTC CTT CTC CTA TAGGC-3′ (SEQ ID NO:7).

[0077] The amplified DNA fragment was digested with SnaBI and NsiI (NsiIsite is generated in the primer DE2A.DBP-start, underlined). Inaddition, a unique BstBI site is generated in this primer (italics).

[0078] Left flanking sequences (corresponding Ad5 nucleotides 21557 to22442):

[0079] DE2A.DBP-stop: 5′-CCA ATG CAT ACG GCG CAG ACG G-3′ (SEQ ID NO:8)

[0080] DE2A.BamHI: 5′-GAG GTG GAT CCC ATG GAC GAG-3′ (SEQ ID NO:9)

[0081] The amplified DNA was digested with BamHI and NsiI (NsiI site isgenerated in the primer DE2A.DBP-stop, underlined). Subsequently, thedigested DNA fragments were ligated into SnaBI/BamHI digestedpBr/Ad.Sal-rITR. Sequencing confirmed the exact replacement of the DBPcoding region with a unique NsiI site and BstBI site in plasmidpBr/Ad.Sal-rITRDE2A. The unique NsiI site and BstBI site can be used tointroduce an expression cassette for a gene to be transduced by therecombinant vector.

[0082] The deletion of the E2A coding sequences was performed such thatthe splice acceptor sites of the 100K encoding L4-gene at position 24048in the top strand was left intact. In addition, the polyadenylationsignals of the original E2A-RNA and L3-RNAs at the left-hand site of theE2A coding sequences were left intact. This ensures proper expression ofthe L3-genes and the gene encoding the 100K L4-protein during theadenovirus life cycle.

[0083] Next, the plasmid pWE/Ad.AflII-rITRDE2A was generated. Theplasmid pBr/Ad.Sal-rITRDE2A was digested with BamHI and Spel. The 3.9-Kbfragment in which the E2A coding region was replaced by the unique NsiIsite and BstBI site was isolated. The pWE/Ad.AflII-rITR was digestedwith BamHil and SpeI. The 35 Kb DNA fragment, from which the BamHI/SpeIfragment containing the E2A coding sequence was removed, was isolated.The fragments were ligated and packaged using 1 phage-packaging extractsaccording to the manufacturer protocol (Stratagene), yielding theplasmid pWE/Ad.AflII-rITRDE2A. Note that there is no sequence overlapbetween the adenoviral sequences present in pWE/Ad.AflII-rITRDE2A andthe E2A sequences present in the expression vectors pcDNA3tsE2A andpcDNAwtE2A or the cell lines derived from this vector.

[0084] I. Generation of the Adapter Plasmids.

[0085] Adapter plasmid pMLP.TK (European patent application no. EP95202213) was modified as follows: SV40 polyA sequences were amplifiedwith primer SV40-1 (introduces a BamHI site) and SV40-2 (introduces aBglII site). In addition, Ad5 sequences present in this construct (fromnt. 2496 to nt. 2779; Ad5 sequences nt. 3511 to 3794) were amplifiedwith primers Ad5-1 (introduces a BglII site) and Ad5-2.

[0086] SV40-1: 5′-GGGGGATCCGAACTTGTTTATTGCAGC-3′ (SEQ ID NO: 10).

[0087] SV40-2: 5′-GGGAGATCTAGACATGATAAGATAC-3′ (SEQ ID NO:11).

[0088] Ad5-1: 5′-GGGAGATCTGTACTGAAATGTGTGGGC-3′ (SEQ ID NO:12).

[0089] Ad5-2:5′-GGAGGCTGCAGTCTCCAACGGCGT-3′ (SEQ ID NO: 13).

[0090] Both PCR fragments were digested with BglII and ligated. Theligation product was amplified with primers SV40-1 and Ad5-2 anddigested with BamHI and AflII. The digested fragment was then ligatedinto pMLP.TK predigested with the same enzymes. The resulting construct,named pMLPI.TK, contains a deletion in adenovirus E1 sequences from nt.459 to nt. 3510.

[0091] This plasmid was used as the starting material to make a newvector in which nucleic acid molecules comprising specific promoter andgene sequences can be easily exchanged. First, a PCR fragment wasgenerated from pZipDMo+PyF101(N⁻) template DNA (described inPCT/NL96/00195) with the following primers: LTR-1: 5′-CTG TAC GTA CCAGTG CAC TGG CCT AGG CAT GGA AAA ATA CAT AAC TG-3′ (SEQ ID NO:14) andLTR-2: 5′-GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGC GTT AACCGG GCG ACT CAG TCA ATC G-3′ (SEQ ID NO: 15). Pwo DNA polymerase(Boehringer Mannheim) was used according to manufacturer's protocol withthe following temperature cycles: once 5 minutes at 95° C.; 3 minutes at55° C.; and 1 minute at 72° C., and 30 cycles of 1 minute at 95° C., 1minute at 60° C., 1 minute at 72° C., followed by once for 10 minutes at72° C. The PCR product was then digested with BamHI and ligated intopMLP10 (Levrero et al., 1991; Gene 101, 195-202) digested with PvuII andBamHI, thereby generating vector pLTR10. This vector contains adenoviralsequences from bp 1 up to bp 454 followed by a promoter consisting ofapart of the Mo-MuLV LTR having its wild-type enhancer sequencesreplaced by the enhancer from a mutant polyoma virus (PyF101). Thepromoter fragment was designated L420. Sequencing confirmed correctamplification of the LTR fragment; however, most 5′ bases in the PCRfragment were missing so that the PvuII site was not restored. Next, thecoding region of the murine HSA gene was inserted. pLTR10 was digestedwith BstBI followed by Klenow treatment and digestion withNcoI. The HSAgene was obtained by PCR amplification on pUC 18-HSA (Kay et al., 1990;J. Immunol. 145, 1952-1959) using the following primers: HSA1, 5′-GCGCCA CCA TGG GCA GAG CGA TGG TGG C-3′ (SEQ ID NO: 16) and HSA2, 5′-GTTAGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTA GAG ATG TAG AA-3′ (SEQID NO:17). The 269 bp-amplified fragment was sub-cloned in a shuttlevector using the NcoI and BglII sites. Sequencing confirmedincorporation of the correct coding sequence of the HSA gene, but withan extra TAG insertion directly following the TAG stop codon. The codingregion of the HSA gene, including the TAG duplication, was then excisedas a NcoI (sticky)-SalI (blunt) fragment and cloned into the 3.5 kb NcoI(sticky)/BstBI (blunt) fragment from pLTR10, resulting in pLTR-HSA10.

[0092] Finally, pLTR-HSA10 was digested with EcoRI and BamHI after whichthe fragment containing the left ITR, packaging signal, L420 promoterand HSA gene was inserted into vector pMLPI.TK digested with the sameenzymes and thereby replacing the promoter and gene sequences. Thisresulted in the new adapter plasmid pAd5/L420-HSA that containsconvenient recognition sites for various restriction enzymes around thepromoter and gene sequences. SnaBI and AvrII can be combined with HpaI,NheI, KpnI, and HindIII to exchange promoter sequences, while the lattersites can be combined with the ClaI or BamHI sites 3′ from HSA codingregion to replace genes in this construct.

[0093] Another adapter plasmid that was designed to allow easy exchangeof nucleic acid molecules was made by replacing the promoter, gene andpolyA sequences in pAd5/L420-HSA with the CMV promoter, a multiplecloning site, an intron and a polyA signal. For this purpose,pAd/L420-HSA was digested with AvrII and BglII followed by treatmentwith Klenow to obtain blunt ends. The 5.1 kb fragment with pBr322 vectorand adenoviral sequences was isolated and ligated to a blunt 1570 bpfragment from pcDNA 1/amp (Invitrogen) obtained by digestion with HhaIand AvrII followed by treatment with T4 DNA polymerase. This adapterplasmid was named pAd5/Clip.

[0094] The adapter plasmid pCMV.LacZ was generated as follows: Theplasmid pCMV.TK (EP 95-202 213) was digested with HindIII, blunted withKlenow and dNTPs and subsequently digested with SalI. The DNA fragmentcontaining the CMV promoter was isolated. The plasmid pMLP.nlsLacZ (EP95-202 213) was digested with KpnI, blunted with T4 DNA polymerase andsubsequently digested with SalI. The DNA fragment containing the LacZgene and adjacent adenoviral sequences was isolated. Next, the two DNAfragments were ligated with T4 DNA ligase in the presence of ATP, givingrise to pCMV.nlsLacZ.

[0095] The adapter plasmid pAd5/CLIP.LacZwas generated as follows: TheE.coli LacZ gene was amplified from the plasmid pMLP.nlsLacZ (EP 95-202213) by PCR with the primers 5′GGGGTGGCCAGGGTACCTCTAGGCTTTTGCAA (SEQ IDNO:18) and 5′GGGGGGATCCATAAACAAGTTCAGAATCC (SEQ ID NO:19). The PCRreaction was performed Ex Taq (Takara) according to the suppliersprotocol at the following amplification program: 5 minutes 94° C., 1cycle; 45 seconds 94° C. and 30 seconds 60° C. and 2 minutes 72° C.,5cycles; 45 seconds 94° C. and 30 seconds 65° C. and 2 minutes 72° C., 25cycles; 10 minutes 72° C., 1 cycle; 45 seconds 94° C. and 30 seconds 60°C. and 2 minutes 72° C., 5 cycles, 1 cycle. The PCR product wassubsequently digested with Kpn1 and BamHI and the digested DNA fragmentwas ligated into KpnI/BamHI digested pcDNA3 (Invitrogen), giving rise topcDNA3.nlsLacZ. Next, the plasmid pAd/CLIP was digested with Spel. Thelarge fragment containing part of the 5′ part CMV promoter and theadenoviral sequences was isolated. The plasmid pcDNA3.nlsLacZ wasdigested with SpeI and the fragment containing the 3′ part of the CMVpromoter and the LacZ gene was isolated. Subsequently, the fragmentswere ligated, giving rise to pAd/CLIP.LacZ. The reconstitution of theCMV promoter was confirmed by restriction digestion.

[0096] The adapter plasmid pAd5/CLIP.Luc was generated as follows: Theplasmid pCMV.Luc (EP 95-202 213) was digested with HindIII and BamHI.The DNA fragment containing the luciferase gene was isolated. Theadapter plasmid pAd/CLIP was digested with HindIII and BamHI, and thelarge fragment was isolated. Next, the isolated DNA fragments wereligated, giving rise to pAdS/CLIP.Luc.

EXAMPLE III

[0097] Generation of Recombinant Adenoviruses

[0098] A. E1-deleted Recombinant Adenoviruses With wt E3 Sequences.

[0099] To generate E1 deleted recombinant adenoviruses with theplasmid-based system, the following constructs are prepared:

[0100] a) An adapter construct containing the expression cassette withthe gene of interest linearized with a restriction enzyme that cuts atthe 3′ side of the overlapping adenoviral genome fragment, preferablynot containing any pBr322 vector sequences, and

[0101] b) A complementing adenoviral genome construct pWE/Ad.AflII-rITRdigested with PacI. These two DNA molecules are further purified byphenol/chloroform extraction and ethanol precipitation. Co-transfectionof these plasmids into an adenovirus packaging cell line, preferably acell line according to the invention, generates recombinant replicationdeficient adenoviruses by a one-step homologous recombination betweenthe adapter and the complementing construct.

[0102] A general protocol as outlined hereinafter and meant as anon-limiting example of the present invention has been performed toproduce several recombinant adenoviruses using various adapter plasmidsand the Ad.AflII-rITR fragment. Adenovirus packaging cells (PER.C6) wereseeded in ˜25 cm² flasks and the next day, when they were at ˜80%confluency, transfected with a mixture of DNA and lipofectamine agent(Life Techn.) as described by the manufacturer. Routinely, 40μllipofectamine, 4 μg adapter plasmid and 4 μg of the complementingadenovirus genome fragment AflII-rITR (or 2 μg of all three plasmids forthe double homologous recombination) are used. Under these conditions,transient transfection efficiencies of ˜50% (48 hrs post transfection)are obtained as determined with control transfections using apAd/CMV-LacZ adapter. Two days later, cells are passaged to ˜80 cm²flasks and further cultured. Approximately five (for the singlehomologous recombination) to eleven days (for the double homologousrecombination) later a cytopathic effect (CPE) is seen, indicating thatfunctional adenovirus has formed. Cells and medium are harvested uponfull CPE and recombinant virus is released by freeze-thawing. An extraamplification step in a 80 cm² flask is routinely performed to increasethe yield since at the initial stage the titers are found to be variabledespite the occurrence of full CPE. After amplification, viruses areharvested and plaque purified on PER.C6 cells. Individual plaques aretested for viruses with active trans-genes.

[0103] Several different recombinant adenoviruses, comprising theluciferase gene (IG.Ad.CLIP.Luc), the bacterial LacZ gene(IG.Ad.CLIP.LacZ and IG.Ad.CMV.LacZ) or an empty CLIP cassette(IG.Ad.CLIP) have been produced using this protocol. In all cases,functional adenovirus was formed and all isolated plaques containedviruses with the expected expression cassettes.

[0104] B. Generation of Recombinant Adenoviruses Deleted for E1 and E2A.

[0105] Besides replacements in the E1 region, it is possible to deleteor replace the E2A region in the adenovirus. This creates theopportunity to use a larger insert or to insert more than one genewithout exceeding the maximum packagable size (approximately 105% of wtgenome length).

[0106] Recombinant viruses that are both E1 and E2A deleted aregenerated by a homologous recombination procedure as described above forE1-replacement vectors using a plasmid-based system consisting of:

[0107] a) An adapter plasmid for E1 replacement according to theinvention, with or without insertion of a first gene of interest.

[0108] b) The pWE/Ad.AflII-rITRDE2A fragment, with or without insertionof a second gene of interest.

[0109] Generation and propagation of such viruses, e.g.,IG.Ad.CMV.LacZDE2A, IG.Ad.CLIP.LacZDE2A, IG.Ad.CLIPDE2A orIG.Ad.CLIP.LucDE2A, requires a complementing cell line forcomplementation of both E1 and E2A proteins in trans, as previouslydescribed herein.

[0110] In addition to replacements in the E1 and E2A region, it is alsopossible to delete or replace (part of) the E3 region in the E1-deletedadenoviral vector, because E3 functions are not necessary for thereplication, packaging and infection of the (recombinant) virus. Thiscreates the opportunity to use larger inserts or to insert more than onegene without exceeding the maximum packagable size (approximately 105%of wt genome length). This can be done, e.g., by deleting part of the E3region in the pBr/Ad.Bam-rITR clone by digestion with XbaI andre-ligation. This removes Ad5 wt sequences 28592-30470 including allknown E3 coding regions. Another example is the precise replacement ofthe coding region of gp19K in the E3 region with a polylinker allowinginsertion of new sequences. This 1) leaves all other coding regionsintact and 2) obviates the need for a heterologous promoter since thetransgene is driven by the E3 promoter and pA sequences, leaving morespace for coding sequences.

[0111] To this end, the 2.7-kb EcoRI fragment from wt Ad5 containing the5′ part of the E3 region was cloned into the EcoRI site of pBluescript(KS-) (Stratagene). Next, the HindIll site in the polylinker was removedby digestion with EcoRV and HincIl and subsequent re-ligation. Theresulting clone pBS.Eco-Eco/ad5DHII was used to delete the gp 19K-codingregion. Primers 1 (5′-GGG TAT TAG GCC AAA GGC GCA-3′ (SEQ ID NO:20)) and2 (5′-GAT CCC ATG GAA GCT TGG GTG GCG ACC CCA GCG-3′ (SEQ ID NO:21))were used to amplify a sequence from pBS.Eco-Eco/ad5DHIII correspondingto sequences 28511 to 28734 in wt Ad5 DNA. Primers 3 (5′-GAT CCC ATG GGGATC CTT TAC TAA GTT ACA AAG CTA-3′ (SEQ ID NO:22)) and 4 (5′-GTC GCT GTAGTT GGA CTG G-3′ (SEQ ID NO:23)) were used on the same DNA to amplifyAd5 sequences from 29217 to 29476. The two resulting PCR fragments wereligated together by virtue of the new introduced NcoI site andsubsequently digested with XbaI and MunI. This fragment was then ligatedinto the pBS.Eco-Eco/ad5DHIII vector that was digested with XbaI(partially) and MunI generating pBS.Eco-Eco/ad5DHIII.Dgp19K. To allowinsertion of foreign genes into the HindIII and BamiHI site, an XbaIdeletion was made in pBS.Eco-Eco/ad5DHIII.Dgp19K to remove the BamHIsite in the Bluescript polylinker. The resulting plasmidpBS.Eco-Eco/ad5DHIII.Dgp19KDXbaI, contains unique HindIII and BamHIsites corresponding to sequences 28733 (HindIII) and 29218 (BamHI) inAd5. After introduction of a foreign gene into these sites, either thedeleted XbaI fragment is re-introduced, or the insert is re-cloned intopBS.Eco-Eco/ad5DHIII.Dgp19K using HindIII and, for example, MunI. Usingthis procedure, we have generated plasmids expressing HSV-TK, hIL-la,rat IL-3, luciferase or LacZ. The unique SrfI and NotI sites in thepBS.Eco-Eco/ad5DHIII.Dgp19K plasmid (with orwithout inserted gene ofinterest) are used to transfer the region comprising the gene ofinterest into the corresponding region of pBr/Ad.Bam-rITR, yieldingconstruct pBr/Ad.Bam-rITRDgp19K (with or without inserted gene ofinterest). This construct is used as described supra to producerecombinant adenoviruses. In the viral context, expression of insertedgenes is driven by the adenovirus E3 promoter.

[0112] Recombinant viruses that are both E1 and E3 deleted are generatedby a double homologous recombination procedure for E1-replacementvectors using a plasmid-based system consisting of:

[0113] a) an adapter plasmid for E1 replacement according to theinvention, with or without insertion of a first gene of interest,

[0114] b) the pWE/Ad.AflII-EcoRI fragment, and

[0115] c) the pBr/Ad.Bam-rITRDgp19K plasmid with or without insertion ofa second gene of interest.

[0116] In addition to manipulations in the E3 region, changes of (partsof) the E4 region can be accomplished easily in pBr/Ad.Bam-rITR.Moreover, combinations of manipulations in the E3 and/or E2A and/or E4region can be made. Generation and propagation of such vectors, however,demands packaging cell lines that complement for E1 and/or E2A and/or E4in trans.

EXAMPLE IV

[0117] E2A Revertant-free Manufacturing of E1/E2A Deleted Vectors onPER.C6/E2A Cells

[0118] The cell lines and E1/E2A deleted vectors described hereinbeforeare developed such that overlap between sequences in the recombinantadenoviral genome and E2A sequences in the complementing cell lines isavoided. This eliminates reversion of the E2A-deleted phenotype in theE1/E2A deleted recombinant adenoviral vectors due to homologousrecombination. The occurrence of reversion of the E2A deleted phenotypewas studied in a PCR assay.

[0119] PER. C6tsE2A clone 3-9 cells were cultured in DMEM supplementedwith 10% FBS and 10 mM MgCl₂in a 10% CO₂ atmosphere at 39° C. in a 25cm² tissue culture flask. At 50% confluency, cells were infected withthe recombinant adenoviral vector IG.Ad.CMV.LacZDE2A and the cells wereput at 32° C. Four days post infection CPE appeared and the cells wereharvested by flicking the flask. Cells were pelleted by centrifugationand the cell pellet was re-suspended in 1 ml/10 mM phosphate buffer (18ml 0.2M Na₂HPO₄ (Baker) and 7 ml 0.2M NaH₂PO₄ (Merck) in 500 ml H₂OpH=7.2). Next, 200 μl 5% sodium deoxycholate (Sigma) was added. Themixture was incubated for 30 minutes at 37° C. and 50 μl 1M MgCl₂ and 10μl DNase (1 MU/ml; ICN) was added. The mixture was incubated for anotherhalf hour at 37° C. and than cleared by centrifugation. The supernatantwas put into a new tube and 100 μl 10% SDS (Baker) and 5 μl Proteinase K(20 mg/ml; Boehringer) were added. The mixture was incubated for 30minutes at 37° C. and subsequently for 15 minutes at 65° C. Next, 1 mlphenol (Sigma) was added and the mixture was tumbled for 1 hour andcentrifuged. One ml of supernatant was put into a fresh tube and 1 mlchloroform (Baker) was added. The mixture was tumbled for another 30minutes and centrifuged. The supernatant was put into a fresh tube andmixed with 1 ml 2-Propanol (Baker) and the DNA was pelleted bycentrifugation. The DNA was washed in 70% Ethanol (Baker) andre-suspended in 200 μl TE and 1 μl RNase (10 mg/ml; Boehringer). The DNAconcentration was determined at a spectrophotometer.

[0120] The recombinant adenoviral vector DNA was screened for reversionof the E2A deleted phenotype by PCR. Two PCR reactions were performed(FIG. 7). The first was a nested PCR reaction for the detection of E2Asequences in the DNA sample. Two primer sets were designed. Set Acontains the primers 551:5′CCGGCAAGTCTTGCGGCATG (SEQ ID NO :24) and 556:5′TAGCAGGTCGGGCGCCGATAT (SEQ ID NO:25) and the nested primers 553:5′GGCTCAGGTGGCTTTTAAGCAG (SEQ ID NO:26) and 554:5′GAGTTGCGATACACAGGGTTGC (SEQ ID NO:27). The PCR reaction was performedusing the eLONGase enzyme mix (Gibco) according to the manufacturer'sprotocol. DNA from 1×10⁹ viral particles (+), which is equivalent to ˜40ng, or water (−) was added as template. The PCR reactions were eithernot spiked, or spiked with 1, 10 and 40 molecules pBR/Ad.Sal-rITR,respectively, as indicated in FIG. 7. The following amplificationprogram for the PCR reaction with primers 551 and 556 was used: 30seconds at 94° C., 1 cycle; 30 seconds 94° C. and 30 seconds at 66° C.and 90 seconds at 68° C., 35 cycles; 10 minutes 68° C., 1 cycle. One μlof this reaction was put into a nested PCR with primers 553 and 554 atthe following amplification program: 30 seconds at 94° C., 1 cycle; 30seconds 94° C. and 30 seconds at 66° C. and 90 seconds at 68° C., 35cycles; 10 minutes 68° C., 1 cycle. This reaction yields a DNA fragmentof 260 bp.

[0121] In the second PCR reaction, a set of primers (Set B) was usedthat flank the E2A gene in the adenoviral genome on the left- and theright-hand site. This PCR reaction amplifies a DNA fragment spanning thesite from which the E2A gene was deleted (FIG. 6). Primer set Bcomprises primer 731 5′AGTGCGCAGATTAGGAGCGC (SEQ ID NO:28) and primer734 5′TCTGCCTATAGGAGAAGGAA (SEQ ID NO:29). The PCR reaction wasperformed using the eLONGase enzyme mix (Gibco) according to themanufacturer protocol. DNA from 1×10⁹ viral particles (+), which isequivalent to ˜40 ng, or water (−) was added as template. The PCRreactions were either not spiked, or spiked with 1, 10 and 40 moleculespBR/Ad.Sal-rITR, respectively, as indicated in FIG. 7. The followingamplification program was used: 30 seconds at 94° C., 1 cycle; 30seconds 94° C. and 30 seconds at 50° C. and 90 seconds at 68° C., 35cycles; 10 minutes 68° C., 1 cycle. This PCR reaction yields a DNAfragment of 169 bp.

[0122] As shown in FIG. 7, left panel (set A), E2A sequences wereamplified from the DNA samples (+) and control samples (−) spiked withboth 1, 10 and 40 molecules, as evidenced by the amplification of a 260bp DNA fragment. In contrast, no E2A sequences were amplified from thenon-spiked samples. This shows that reversion of the E2A-deleted doesnot occur. The PCR reactions with primers 731/734 yielded the expectedDNA fragment of 169 bp in the samples containing the recombinantadenoviral vector DNA (+). From the negative control samples containingthe water instead of DNA (−), no DNA fragment of 169 bp was amplified.These data show that elimination of overlap between adenoviral sequencesin the vector and cell line prevents reversion of the E2A-deletedphenotype.

EXAMPLE V

[0123] Transduction Capacity of and Residual Expression of AdenoviralGenes from E1-deleted and E1/E2A-deleted Recombinant Adenoviral Vectors

[0124] The transduction capacity of E1/E2A deleted vectors was comparedto E1 deleted vectors. HeLa cells were seeded at 5×10⁵ cells/well in 6well plates (Greiner) in DMEM supplemented with 10% FBS in a 10% CO₂atmosphere at 37° C. The next day, cells were infected with a m.o.i. ofeither 0, 10, 100 or 1000 viral particles IG.Ad/CMV.LacZ orIG.Ad/CMV.LacZDE2A per cell. Forty-eight hours post infection, cellswere washed with PBS (NPBI) and fixed for 8 minutes in 0.25%glutaraldehyde (Sigma) in PBS (NPBI). Subsequently, the cells werewashed twice with PBS and stained for 8 hours with X-gal solution (1mg/ml X-gal in DMSO (Gibco), 2 mM MgCl₂ (Merck), 5 mM K₄[Fe(CN)₆].3H₂O(Merck), 5mM K₃[Fe(CN)₆] (Merck) in PBS. The reaction was stopped byremoval of the X-gal solution and washing of the cells with PBS. FIG. 8shows that IG.Ad/CMV.LacZDE2A transduced HeLa cells at least as good asdid IG.Ad/CMV.LacZ at all m.o.i.'s. Comparable results were obtainedafter infection of IG.Ad.CLIP.LacZ and IG.Ad.CLIP.LacZDE2A and afterinfection of A549 cells with the respective recombinant adenoviralvectors. These data show that the viral particle to transduction unitratio (vp/tu) of E1/E2A deleted vectors (e.g., IG.Ad/CMV.LacZDE2A) is atleast as good as the vp/tu of E1 deleted vectors (e.g., IG.Ad/CMV.LacZ).

[0125] Next, the vp/tu ratio of E1- and E1/E2A-deleted vectors wasdetermined in a more sensitive assay, i.e., a luciferase assay. HeLa andA549 cells were seeded at 5×10⁵ cells/well in 6 well plates (Greiner) inDMEM supplemented with 10% FBS in a 10% CO₂ atmosphere at 37° C. Thenext day, cells were infected with a m.o.i. of either 0, 10, 100, 1,000or 10,000 vp/cell IG.Ad/CLIP.Luc or IG.Ad/CLIP.LucDE2A per cell. Twodays post infection, cells were lysed and the luciferase activity wasdetermined with the Luciferase Assay System according to the protocol ofthe supplier (Promega). FIG. 9 shows that both the IG.Ad/CLIP.LucDE2Ainfected HeLa and A549 cells produce more luciferase enzyme than theIG.Ad/CLIP.Luc infected HeLa and A549 and HeLa cells, at all m.o.i.'stested. These data confirm that E1/E2A deleted recombinant adenoviralvectors produced on PER.C6ts125E2A cells have a vp/tu ratio that is atleast as good as the vp/tu ratio of E1 deleted vectors. The above is incontrast to what has recently been reported by others (O'Neal et al.,1998; Lusky et al., 1998), who found that the vp/tu ratio of E1/E2Adeleted recombinant adenoviral vectors is impaired significantly.However, these vectors were produced on two independent 293 based E2Acomplementing cell lines harboring inducible E2A genes. This suggeststhat the use of temperature sensitive E2A genes, such as ts125E2A,yields superior E2A complementing cell lines as compared to the commonlyused inducible E2A genes.

[0126] In order to test whether E1/E2A deleted vectors residuallyexpress adenoviral proteins, the following experiment has beenperformed. A549 cells were seeded on 6 well plates (Greiner) at adensity of 5×10⁵ cells/well in DMEM supplemented with 10% FBS in a 10%CO₂ atmosphere at 37° C. The next day, cells were infected with a m.o.i.of either 0, 100, 1,000 or 10,000 vp/cell IG.Ad/CLIP or IG.Ad.CLIPDE2A.After 12 hours, the infection medium was replaced by fresh DMEMsupplemented with 10% FBS. Seventy-two hours post infection, the cellswere washed with PBS (NPBI) and lysed and scraped in RIPA (1% NP-40,0.5% sodium deoxycholate and 0.1% SDS in PBS, supplemented with 1 mMphenylmethylsulfonylfluoride and 0.1 mg/ml trypsin inhibitor). After 15minutes incubation on ice, the lysates were cleared by centrifugation.Protein concentrations were determined by the BioRad protein assay,according to standard procedures of the supplier (BioRad). Equal amountsof whole-cell extract were fractionated by SDS-PAGE on 10% gels intriplicate. Proteins were transferred onto Immobilon-P membranes(Millipore) and incubated with the aDBP monoclonal antibody B6, thepolyclonal a-Penton base antibody Ad2-Pb571 (kind gift of Dr. P.Boulanger, Montpellier, France) and the polyclonal a-knob domainantibody of fiber E641/3 (kind gift of R. Gerard, Leuven, Belgium). Thesecondary antibodies were ahorseradish-peroxidase-conjugated goat antimouse and ahorseradish-peroxidase-conjugated goat anti rabbit (BioRad).The Western blotting procedure and incubations were performed accordingto the protocol provided by Millipore. The complexes were visualizedwith the ECL detection system according to the manufacturer's protocol(Amersham). FIG. 10 shows that cells infected with IG.Ad.CLIP expressboth E2A encoded DBP, Penton base and Fiber proteins. The proteinsco-migrated with the respective proteins in the positive control (laneP, extract from PER.C6 cells infected with IG.Ad.CLIP harvested atstarting CPE). The residual expression of these proteins in A549 cellswas m.o.i. dependent. In contrast, no DBP, penton-base or fiber wasdetected in the non-infected A549 cells or cells infected withIG.Ad.CLIPDE2A. These data show that deletion of the E2A gene did notonly eliminate residual DBP expression, but also the residual expressionof the late adenoviral proteins, penton-base and fiber.

[0127] In conclusion, the foregoing shows that E1/E2A deleted vectorsproduced on PER.C6/tsE2A complementing cell lines have a favorablephenotype. First, these vectors have an ideal vp/tu ratio, which is atleast as good as that of E1 deleted vectors. Second, the E1/E2A deletedvectors do not residually express detectable amounts of E2A encoded DBPor late gene encoded penton-base or fiber. This favorable phenotypeimproves the prospects for the use of recombinant adenoviral vectors ingene therapy.

1 29 1 35 DNA Artificial Sequence Description of Artificial Sequenceprimer DBPpcr1 1 cgggatccgc caccatggcc agtcgggaag aggag 35 2 33 DNAArtificial Sequence Description of Artificial Sequence primer DBPpcr2 2cggaattctt aaaaatcaaa ggggttctgc cgc 33 3 23 DNA Artificial SequenceDescription of Artificial Sequence oligo-linker containing PacI site 3aattgtctta attaaccgct taa 23 4 19 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide used to form oligo-linker describedby SEQ. ID. NO. 3 4 aattgtctta attaaccgc 19 5 19 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide used to formoligo-linker described by SEQ. ID. NO. 3 5 aattgcggtt aattaagac 19 6 23DNA Artificial Sequence Description of Artificial Sequence primerDE2A.SnaBI 6 ggcgtacgta gccctgtcga aag 23 7 35 DNA Artificial SequenceDescription of Artificial Sequence primer DE2A.DBP-start 7 ccaatgcattcgaagtactt ccttctccta taggc 35 8 22 DNA Artificial Sequence Descriptionof Artificial Sequence primer DE2A.DBP-stop 8 ccaatgcata cggcgcagac gg22 9 21 DNA Artificial Sequence Description of Artificial Sequenceprimer DE2A.BamHI 9 gaggtggatc ccatggacga g 21 10 27 DNA ArtificialSequence Description of Artificial Sequence primer SV40-1 10 gggggatccgaacttgttta ttgcagc 27 11 25 DNA Artificial Sequence Description ofArtificial Sequence primer SV40-2 11 gggagatcta gacatgataa gatac 25 1227 DNA Artificial Sequence Description of Artificial Sequence primerAd5-1 12 gggagatctg tactgaaatg tgtgggc 27 13 24 DNA Artificial SequenceDescription of Artificial Sequence primer Ad5-2 13 ggaggctgca gtctccaacggcgt 24 14 47 DNA Artificial Sequence Description of Artificial Sequenceprimer LTR-1 14 ctgtacgtac cagtgcactg gcctaggcat ggaaaaatac ataactg 4715 63 DNA Artificial Sequence Description of Artificial Sequence primerLTR-2 15 gcggatcctt cgaaccatgg taagcttggt accgctagcg ttaaccgggcgactcagtca 60 atc 63 16 27 DNA Artificial Sequence Description ofArtificial Sequence primer HSA1 16 gcgccaccat gggcagagcg atggtgg 27 1750 DNA Artificial Sequence Description of Artificial Sequence primerHSA2 17 gttagatcta agcttgtcga catcgatcta ctaacagtag agatgtagaa 50 18 32DNA Artificial Sequence Description of Artificial Sequence primer usedfor amplification of E. coli LacZ 18 ggggtggcca gggtacctct aggcttttgc aa32 19 29 DNA Artificial Sequence Description of Artificial Sequenceprimer used for amplification of E. coli LacZ 19 ggggggatcc ataaacaagttcagaatcc 29 20 20 DNA Artificial Sequence Description of ArtificialSequence primer 1 20 gggtattagg ccaaaggcgc 20 21 33 DNA ArtificialSequence Description of Artificial Sequence primer 2 21 gatcccatggaagcttgggt ggcgacccca gcg 33 22 36 DNA Artificial Sequence Descriptionof Artificial Sequence primer 3 22 gatcccatgg ggatccttta ctaagttacaaagcta 36 23 19 DNA Artificial Sequence Description of ArtificialSequence primer 4 23 gtcgctgtag ttggactgg 19 24 20 DNA ArtificialSequence Description of Artificial Sequence primer 551 24 ccggcaagtcttgcggcatg 20 25 21 DNA Artificial Sequence Description of ArtificialSequence primer 556 25 tagcaggtcg ggcgccgata t 21 26 22 DNA ArtificialSequence Description of Artificial Sequence primer 553 26 ggctcaggtggcttttaagc ag 22 27 22 DNA Artificial Sequence Description of ArtificialSequence primer 554 27 gagttgcgat acacagggtt gc 22 28 20 DNA ArtificialSequence Description of Artificial Sequence primer 731 28 agtgcgcagattaggagcgc 20 29 20 DNA Artificial Sequence Description of ArtificialSequence primer 734 29 tctgcctata ggagaaggaa 20

What is claimed is:
 1. A cell capable of at least in part complementingadenovirus E2A function of an adenovirus defective in E2A function, saidcell comprising a nucleic acid encoding adenovirus E2A or a functionalpart thereof, derivative thereof, analogue thereof, or mixture of any ofthese.
 2. The cell of claim 1 wherein said nucleic acid encodingadenovirus E2A is a temperature sensitive adenovirus.
 3. The cell ofclaim 2 wherein said nucleic acid encoding adenovirus E2A is ofadenovirus ts125 origin.
 4. The cell of claim 1, claim 2, or claim 3further comprising a nucleic acid encoding adenovirus E1-region proteinsor a functional part, derivative thereof, analogue thereof, or mixtureof any thereof.
 5. The cell of claim 4 wherein said cell is of PER.C6(ECACC deposit number 96022940) origin.
 6. A method for producing anadenoviral particle containing an adenovirus vector with a functionaldeletion of E2A, said method comprising: providing a cell according toclaim 1, claim 2, claim 3, claim 4, or claim 5 with said adenovirusvector, culturing said cell, and harvesting said adenoviral particle. 7.The method for producing an adenoviral particle containing an adenovirusvector with a functional deletion of E2A according to claim 6 whereinsaid functional deletion comprises a deletion of at least part of thenucleic acid encoding E2A.
 8. The method for producing an adenoviralparticle containing an adenovirus vector with a functional deletion ofE2A according to claim 6 wherein said nucleic acid encoding adenovirusE2A in said cell's genome does not comprise sequence overlap with saidvector which leads to replication competent adenovirus and/or to theformation of an adenovirus vector comprising E2A function.
 9. The methodfor producing an adenoviral particle according to claim 6, said methodfurther comprising: providing an adenovirus vector further comprising afunctional deletion of E1-region encoding nucleic acid said adenovirusvector to said cell, said cell further characterized in: being capableof at least in part complementing adenovirus E2A function of anadenovirus defective in E2A function, comprising a nucleic acid encodingadenovirus E2A or a functional part thereof, derivative thereof, and/oranalogue thereof, and further comprising a nucleic acid sequenceencoding adenovirus E1-region proteins or a functional part, derivativethereof, and/or analogue thereof, culturing said cell, and harvestingsaid virus particle.
 10. The method according to claim 9 wherein saidnucleic acid encoding adenovirus E1-region has no sequence overlap withsaid vector which leads to replication competent adenovirus and/or tothe formation of an adenovirus vector comprising an E1 function.
 11. Amethod according to claim 6, claim 7, claim 8, claim 9, or claim 10wherein said adenovirus vector further comprises at least one nucleicacid of interest.
 12. An adenovirus vector comprising a functionaldeletion of adenovirus E2A.
 13. The adenovirus vector of claim 12wherein said vector comprises a deletion of at least part of the nucleicacid encoding E2A.
 14. The adenovirus vector of claim 13 wherein saiddeletion encompasses at least the entire coding region of E2A.
 15. Theadenovirus vector of claim 14 further comprising a deletioncorresponding to a deletion of nucleotides 22443 to 24032 in adenovirus5.
 16. The adenovirus vector of claim 12, claim 13, claim 14, or claim15 further comprising a deletion of nucleic acid encoding E1-regionproteins or parts, derivatives and/or analogues thereof.
 17. Theadenovirus vector of claim 12, claim 13, claim 14, claim 15, or claim 16wherein said deletion of nucleic acid encoding E1-region proteinscomprises a deletion corresponding to a deletion of nucleotides 459 to3510 in adenovirus
 5. 18. The adenovirus vector of claim 12, claim 13,claim 14, claim 15, claim 16, or claim 17 further comprising at leastone nucleic acid of interest.
 19. A preparation of adenovirus vectorcontaining adenovirus particles wherein said adenovirus vector comprisesa functional deletion of E2A.
 20. The preparation of claim 19 whereinsaid adenovirus vector further comprises a deletion of nucleic acidencoding E1-region proteins or parts, derivatives and/or analoguesthereof.
 21. The preparation of claim 19 or claim 20 free of adenovirusvectors comprising E2A function.
 22. The preparation of claim 21, saidpreparation characterized in being free of adenovirus vectors comprisingnucleic acid encoding a functional E2A, or a functional part, derivativeand/or analogue thereof.
 23. The preparation according to claim 21 orclaim 22 free of adenovirus vectors comprising nucleic acid encodingE1-region proteins or parts, derivatives and/or analogues thereof.
 24. Amethod for providing cells of an individual with a nucleic acid ofinterest, without risk of administering simultaneously a replicationcompetent adenovirus vector, comprising administering said individual apreparation according to claim 19, claim 20, claim 21, claim 22, orclaim
 23. 25. The method of claim 24 wherein said preparation ischaracterized in being free of adenovirus vectors comprising nucleicacid encoding a functional E2A, or a functional part, derivative and/oranalogue thereof.
 26. The method of claim 24 or claim 25 wherein saidpreparation is free of adenovirus vectors comprising nucleic acidencoding E1-region proteins or parts, derivatives and/or analoguesthereof.
 27. An adenovirus vector comprising at least a deletion of aregion which in adenovirus 5 corresponds to nucleotides 22443-24032. 28.An adenovirus vector comprising at least a deletion of a region which inadenovirus 5 corresponds to nucleotides 22418-24037.
 29. An adenovirusvector comprising at least a deletion of a region which in adenovirus 5corresponds to nucleotides 22348-24060.
 30. An adenovirus vectoraccording to claim 27, claim 28, or claim 29 further comprising atleastnucleic acidwhichin adenovirus 5 corresponds to nucleotides3534-22347 and/ornucleotides 24061 until the right ITR.
 31. Anadenovirus vector according to claim 27 or claim 28 further comprisingat least nucleic acid which in adenovirus 5 corresponds to nucleotides3534-22417 and/ornucleotides 24038 until the right ITR.
 32. Anadenovirus vector according to claim 27 further comprising at leastnucleic acid which in adenovirus 5 corresponds to nucleotides 3534-22442and/or nucleotides 24033 until the right ITR.
 33. An adenovirus vectoraccording to claim 27 further comprising at least nucleic acid which inadenovirus 5 corresponds to nucleotides 3534-22442 and/or nucleotides24033 until the right ITR.
 34. The cell of claim 1 wherein said nucleicacid is integrated into said cell's genome.
 35. The cell of claim 4wherein said cell is derived from cell line
 293. 36. The methodaccording to claim 9 wherein said nucleic acid is integrated into saidcell's genome.
 37. A cell capable of at least in part complementingadenovirus E2A function of an adenovirus defective in E2A function, saidcell comprising a nucleic acid encoding adenovirus E2A or a functionalpart thereof.
 38. The cell of claim 37, said cell further comprising anucleic acid encoding adenovirus E1-region proteins or a functional partthereof.
 39. The cell of claim 38, wherein said nucleic acid encodingadenovirus E2A encodes a temperature sensitive E2A mutant.
 40. The cellof claim 39, wherein said temperature sensitive E2A mutant is an E2Amutant encoded by adenovirus ts125.
 41. The cell of claim 38, whereinsaid cell is of cell line 293 origin.
 42. The cell of claim 39, whereinsaid cell is of cell line 293 origin.
 43. The cell of claim 40, whereinsaid cell is of cell line 293 origin.
 44. A method for producing anadenoviral particle containing an adenovirus vector with a functionaldeletion of E2A, said method comprising: providing a cell according toany one of claims 37-43 with said adenovirus vector, culturing saidcell, and harvesting said adenoviral particle.
 45. The method accordingto claim 44, wherein said functional deletion comprises a deletion of atleast part of the nucleic acid encoding E2A.
 46. The method according toclaim 45, wherein said nucleic acid encoding adenovirus E2A in saidcell's genome does not comprise sequence overlap with said vector whichleads to replication competent adenovirus and/or to the formation of anadenovirus vector comprising E2A function.
 47. The method according toclaim 45, said method further comprising: providing an adenovirus vectorfurther comprising a functional deletion of E1-region encoding nucleicacid said adenovirus vector to said cell, said cell furthercharacterized by: being capable of at least in part complementingadenovirus E2A function of an adenovirus defective in E2A function,comprising a nucleic acid encoding adenovirus E2A or a functional partthereof, derivative thereof, and/or analogue thereof, and furthercomprising a nucleic acid sequence encoding adenovirus E1-regionproteins or a functional part, derivative thereof, and/or analoguethereof, culturing said cell, and harvesting said virus particle.
 48. Anadenovirus vector comprising a functional deletion of adenovirus E2A.49. The adenovirus vector of claim 48 wherein said vector comprises adeletion of at least part of the nucleic acid encoding E2A.
 50. Theadenovirus vector of claim 49 wherein said deletion encompasses at leastthe entire coding region of E2A.
 51. The adenovirus vector of claim 48,further having a deletion of nucleic acid encoding E1-region proteins orparts, derivatives and/or analogues thereof.
 52. The adenovirus vectorof claim 49, further having a deletion of nucleic acid encodingE1-region proteins or parts, derivatives and/or analogues thereof. 53.The adenovirus vector of claim 50 further having a deletion of nucleicacid encoding E1-region proteins or parts, derivatives and/or analoguesthereof.
 54. A preparation of adenovirus vector containing adenovirusparticles wherein said adenovirus vector has a functional deletion ofE2A.
 55. The preparation of claim 54 wherein said adenovirus vectorfurther has a deletion of nucleic acid encoding E1-region proteins orparts, derivatives and/or analogues thereof.
 56. The preparation ofclaim 54 free of adenovirus vectors comprising E2A function.
 57. Thepreparation of claim 55 free of adenovirus vectors comprising E2Afunction.
 58. The preparation of claim 56, said preparationcharacterized in being free of adenovirus vectors comprising nucleicacid encoding a functional E2A, or a functional part, derivative and/oranalogue thereof.
 59. The preparation of claim 57, said preparationcharacterized in being free of adenovirus vectors comprising nucleicacid encoding a functional E2A, or a functional part, derivative and/oranalogue thereof.
 60. The preparation of claim 56, free of adenovirusvectors comprising nucleic acid encoding E1-region proteins or parts,derivatives and/or analogues thereof.
 61. The preparation of claim 57,free of adenovirus vectors comprising nucleic acid encoding E1-regionproteins or parts, derivatives and/or analogues thereof.
 62. Thepreparation of claim 58, free of adenovirus vectors comprising nucleicacid encoding E1-region proteins or parts, derivatives and/or analoguesthereof.
 63. The preparation of claim 59 free of adenovirus vectorscomprising nucleic acid encoding E1-region proteins or parts,derivatives and/or analogues thereof.
 64. A method for providing cellsof a subject with a nucleic acid of interest, with a decreased risk ofadministering simultaneously a replication competent adenovirus vector,said method comprising administering to the subject's cells thepreparation of any one of claims 54-63.
 65. An adenovirus vectorcomprising at least a deletion of a region which in adenovirus 5corresponds to nucleotides 22443-24032, or nucleotides 22418-24037, ornucleotides 22348-24060, further comprising at least nucleic acid whichin adenovirus 5 corresponds to nucleotides 3534-22347 and/or nucleotides24061 until the right ITR.
 66. The method according to claim 44, whereinsaid nucleic acid is integrated into said cell's genome.
 67. The methodaccording to claim 45, wherein said nucleic acid is integrated into saidcell's genome.
 68. The method according to of claim 46, wherein saidnucleic acid is integrated into said cell's genome.
 69. The methodaccording to claim 47, wherein said nucleic acid is integrated into saidcell's genome.